Project Abstract The goal of this Phase I SBIR is to develop a new platform that enables dehydrogenases to be used as enzymes in amperometric biosensors. Regardless of the platform or measurement modality, all biosensor designs require the use of an enzyme to properly function. Biosensors are devices that measure compounds important for human health. FDA approval has already been achieved for amperometric biosensors that monitor glucose in humans, and several new biosensor platforms are currently under development. Biosensors require an enzyme that is specific for the compound to be measured. This proposal will showcase our platform?s utility by developing an enzyme system that provides the ability to monitor cortisol in point-of-care devices and biosensors. The major impediment to the development of a continuous cortisol monitoring system is the lack of an enzyme that can be used as part of device fabrication. At this time, no enzyme suitable for cortisol detection and suitable for biosensing applications has been described or commercialized. This illustrates the importance of developing new enzyme systems specific for analytes important in human health. Once proof-of-concept is demonstrated in Phase I, our Phase II will extend the platform for use with other dehydrogenases to monitor analytes important in human health and disease. Phase II targets include maleate dehydrogenase, GABA dehydrogenase, succinate dehydrogenase, pyruvate dehydrogenase, acetaldehyde dehydrogenase, and alcohol dehydrogenase. These dehydrogenase enzymes either have no known oxidase counterpart or have oxidase counterparts that are so unstable as to render the enzyme essentially useless in biosensor fabrication for studies in freely moving animals. At the end of the Phase II SBIR, we will have an optimized platform that produces highly stabilized dehydrogenase enzymes for use in biosensors. In addition, a functioning cortisol biosensor suitable for use in humans will be developed. No diagnostic device presently exists to continuously measure cortisol level in vivo. In this study we will use our proposed platform to create a cortisol enzyme system by coupling together two novel enzymes to produce a signal that can be detected on an electrode with high sensitivity. This dual enzyme approach that features a covalently attached co-enzyme molecule, is highly innovative and will enable, for the first time, the continuous monitoring analytes important to human health such as cortisol. New devices based on this technology will allow clinicians to monitor and manage pain, stress, and other conditions that negatively impact human health. Our platform will positively impact the development and manufacturing of other devices for novel sensing applications. The Phase I deliverable will be a prototype of the coupled enzyme system demonstrating proof-of-concept for a selective, sensitive and efficient mechanism of monitoring cortisol levels in tissues and fluids. The successful completion of this proposal is the necessary first step to establishing a continuous method of monitoring cortisol in all at risk individuals.